Fetal calf serum-free culture of Chinese hamster ovary cells employing fish serum

The effects of fish serum on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of Chinese hamster ovary (CHO) cells DR1000L4N were investigated and compared with those of fetal calf serum (FCS). Although fish serum did not stimulate the initial adhesion of CHO cells to culture dishes, it prompted cell growth after cell adhesion with FCS for 24 h. The cell density in the fish serum medium reached 75% that in the FCS medium. Fish serum promoted cell adhesion to and cell growth on collagen-coated dishes. The cell-specific production rate of hGM-CSF in the fish serum medium on collagen-coated dishes was almost the same as that in the FCS medium.     

Growth inhibitory effects of dimethyl sulfoxide and dimethyl sulfone on vascular smooth muscle and endothelial cells in vitro

Summary The growth of bovine aortic smooth muscle and endothelial cells was studied after exposure to dimethyl sulfoxide (DMSO) or its major metabolite, dimethyl sulfone (DMSO₂). Both compounds caused a dose-dependent inhibition of cell growth as determined by [³H]thymidine incorporation and by counting the number of cells with time of exposure in culture. The IC₅₀ of DMSO (concentration which produces 50% inhibition of growth) was 1% for smooth muscle cells and 2.9% for endothelial cells. Similarly, the IC₅₀ of DMSO₂ was also 1% for smooth muscle cells, but was 1.8% for endothelial cells. After a 4-d exposure to either compound, the growth inhibition of smooth muscle cells was completely reversible at 1%, partially reversible at 2 to 3% and completely irreversible at 4%. By comparison, inhibition of endothelial cell growth was completely reversible up to 4% of either compound. It is concluded that the growth of smooth muscle cells was similarly inhibited by DMSO, and DMSO₂, but that smooth muscle cells were more susceptible than endothelial cells to the growth inhibitory effects of these compounds. In addition, DMSO₂ was a more potent inhibitor of cell growth than DMSO and its growth inhibition was less reversible than that produced by DMSO.     

Effects of synthetic lysophosphatidylcholines on suspension cultures of the Chinese hamster ovary DG44 cells in protein-free media

This study described the effects of synthetic lysophosphatidylcholines on the growth of recombinant CHO-DG44 cells in suspension. Overall, cell growth characteristics were improved when cultivated in suspension in a protein-free medium supplemented with natural soybean lysophosphatidylcholines. To substitute synthetic lysophosphatidylcholines for the naturally occurring lysophosphatidylcholines, we implemented a systematic approach in which twelve synthetic lysophosphatidylcholines were grouped into three lipid mixtures according to the length of their acyl chains. We found that synthetic lysophosphatidylcholines with medium acyl chain lengths (C14-C18), including oleoyl lysophosphatidylcholine (C18:1) could increase cell growth in the protein-free media. The fortified protein-free medium with medium acyl chain length lysophosphatidylcholines (C14-C18) maintained growth of CHO-DG44 cells over five consecutive passages, whereas the cell growth in a CHO protein-free medium was decreased gradually after four passages. We also observed that the restorative effect of oleoyl lysophosphatidylcholine was comparable to that of natural lysophosphatidylcholine in batch and long-term cultivation. These results show that synthetic lysophosphatidylcholines can be used as lipid supplements in either protein-free media or chemically defined media for CHO cell suspension cultures.     

Effects of heat treatment and concentration of fish serum on cell growth in adhesion culture of Chinese hamster ovary cells

The effects of heat treatment and concentration of fish serum (FS) on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of recombinant Chinese hamster ovary (CHO) cells, DR1000L4N, were investigated. The addition of heat treated FS instead of non-heat-treated FS improved cell growth in terms of cell density, which reached 60% that in 10% fetal calf serum (FCS)-containing medium (FCS medium). A decrease in FS concentration from 10 to 1.25% markedly increased cell density, which was 79% that in 10% FCS medium. The combination of heat treatment at 56 °C and the addition of FS at a low concentration (1.25%) showed an additive effect on cell growth and resulted in the same cell density as that in 10% FCS medium, whereas the hGM-CSF concentration in the culture using FS-containing medium (FS medium) was approximately 50% that in 10% FCS medium. The total lipid concentration in FS was more than three fold that in FCS. The effect of decreasing FS concentration on cell growth may be due to the low lipid concentration in FS medium, because addition of the lipids extracted from FS to 10% FCS and 1.25% FS media markedly decreased cell density. Consequently, the addition of heat-treated FS at low concentrations to medium may be useful for the growth of CHO cells without FCS.     

Effects of cysteine,asparagine, or glutamine limitations in Chinese hamster ovary cell batch and fed-batch cultures

Amino acid availability is a key factor that can be controlled to optimize the productivity of fed-batch cultures. To study amino acid limitation effects, a serum-free chemically defined basal medium was formulated to exclude the amino acids that became depleted in batch culture. The effect of limiting glutamine, asparagine, and cysteine on the cell growth, metabolism, antibody productivity, and product glycosylation was investigated in three Chinese hamster ovary (CHO) cell lines (CHO-DXB11, CHO-K1SV, and CHO-S). Cysteine limitation was detrimental to both cell proliferation and productivity for all three CHO cell lines. Glutamine limitation reduced growth but not cell specific productivity, whereas asparagine limitation had no significant effect on either growth or cell specific productivity. Neither glutamine nor asparagine limitation significantly affected antibody glycosylation. Replenishing the CHO-DXB11 culture with cysteine after 1 day of cysteine limitation allowed the cells to partially recover their growth and productivity. This recovery was not observed after 2 days of cysteine limitation. Based on these findings, a fed-batch protocol was developed using single or mixed amino acid supplementation. Although cell density and antibody concentration were lower compared to a commercial feed, the feeds based on cysteine supplementation yielded comparable cell specific productivity. Overall, this study showed that different amino acid limitations have varied effects on the performance of CHO cell cultures and that maintaining cysteine availability is a critical process parameter for the three cell lines investigated.     

Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line

A proteomics approach was used to identify the proteins potentially implicated in the cellular response concomitant with elevated production levels of human growth hormone in a recombinant Chinese hamster ovary (CHO) cell line following exposure to 0.5 mM butyrate and 80 microM zinc sulphate in the production media. This involved incorporation of two-dimensional (2-D) gel electrophoresis and protein identification by a combination of N-terminal sequencing, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, amino acid analysis and cross species database matching. From these identifications a CHO 2-D reference map and annotated database have been established. Metabolic labelling and subsequent autoradiography showed the induction of a number of cellular proteins in response to the media additives butyrate and zinc sulphate. These were identified as GRP75, enolase and thioredoxin. The chaperone proteins GRP78, HSP90, GRP94 and HSP70 were not up-regulated under these conditions.     

Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.     

Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults

Apoptosis has been found to occur in bioreactors as a result of environmental stresses. The overexpression of bcl-2 is a widely used strategy to limit the induction of apoptosis in mammalian cell cultures. In this study, the effectiveness of wild-type Bcl-2 was compared to a Bcl-2 mutant lacking the nonstructured loop domain in two commercially prominent cell lines, Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells. The generation of a DNA "ladder" and condensation of chromatin indicated that apoptosis occurred in these cell lines following Sindbis virus infection and serum deprivation. When cells were engineered to overexpress the bcl-2 mutant, cell death due to Sindbis virus was inhibited in a concentration-dependent manner. Furthermore, the Bcl-2 mutant provided increased protection as compared to wild-type Bcl-2 following two model insults, Sindbis virus infection and serum deprivation. Total production for a heterologous protein encoded on the Sindbis virus was increased in cell lines expressing the Bcl-2 variants compared to the parental cell line. In order to understand the reasons for the improved anti-apoptosis properties of the mutant, wild-type Bcl-2 and mutant Bcl-2 were examined by Western blot following each model insult. Wild-type Bcl-2 was observed to degrade into a 23 kDa fragment following both Sindbis virus infection and serum withdrawal in both cell lines, while the mutant Bcl-2 protein was not degraded during the same period. The processing of Bcl-2 was found to correlate with reduced cell viabilities following the two external insults to suggest that Bcl-2 degradation may limit its ability to inhibit apoptosis. These studies indicate that the cells regulate anti-apoptosis protein levels and these processing events can limit the effectiveness of cell death inhibition strategies in mammalian cell culture systems.     

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500?µg/L) and the half-life of IGF-1 in blood circulation is only 4.5?min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA–IGF-1 reached 100?mg/L. The fusion protein HSA–IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA–IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA–IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA–IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.     

Segmented linear modeling of CHO fed‐batch culture and its application to large scale production

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed‐batch cultures. Using the model structure and parameter values from a small‐scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed‐batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785–797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

The effect of dimethyl sulfoxide on the function of cytochrome P450 2D6 in HepG2 cells upon the co-expression with NADPH-cytochrome P450 reductase

HepG2 cells, a human hepatoma cell line, stably expressing NADPH-cytochrome P450 reductase (OR) and/or cytochrome P450 2D6 wild-type (CYP2D6-WT) or its variants (Pro34Ser, Gly42Arg, Arg296Cys and Ser486Thr) were established in the present study. The cultivation of HepG2 cells expressing CYP2D6-WT in the culture medium containing dimethyl sulfoxide (DMSO, 0.1% of final concentration) markedly increased the bufuralol (BF) 1'-hydroxylase activity compared with that of control cells when cultivated without DMSO. A similar effect was also observed in HepG2 cells stably expressing CYP2D6 and OR. The addition of hemin in place of DMSO to the culture medium resulted in no increase in the enzyme activity. Western blot analysis revealed that the levels of CYP2D6 protein were similar between DMSO-treated and non-treated HepG2 cells regardless of OR expression. Spectrophotometric analysis of reduced carbon monoxide-difference spectra of HepG2 cells expressing CYP2D6-WT and/or OR demonstrated that the addition of DMSO increased the peak height of functional CYP2D6 at 450 nm. These results suggest that the increase in CYP2D6 activity is attributable to the radical-scavenging effect of DMSO. The HepG2 cell lines stably expressing OR and CYP2D6 or its variants in combination with DMSO treatment may be useful for screening the cytotoxicity of chemical compounds which undergo oxidation by CYP2D6.     

Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases

Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance.     

Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: Cloning efficiency,growth in suspension,and selection of drug-resistant mutant phenotypes

Summary Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 μg/ml insulin, 3×10⁻⁷ M linoleic acid, 1×10⁻⁸ M H₂SeO₃) or bovine serum albumin (BSA, 1% wt/vol). With the additives and ≤0.5% fetal bovine serum (FBS), Ham’s F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle’s minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham’s F12, 1% FBS+deoxycytidine+BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at theaprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethyl-nitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels. This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-ENG-48 and supported by the Environmental Protection Agency under Interagency Agreements IAG-D5-E681-AN and AO.     

Evaluating the interaction between UCOE and DHFR‐linked amplification and stability of recombinant protein expression

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry. In the creation of mammalian cell lines plasmid DNA carrying the gene‐of‐interest integrates randomly into the host cell genome, which results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long‐term culture. This means that a large number of clones need to be screened in order to isolate stable, high producing cell lines making mammalian cell line development a long and laborious process. In this study an expression platform incorporating a Ubiquitous Chromatin Opening Element (UCOE; which are proposed to maintain chromatin in an open state) has been utilised for the expression of eGFP in CHO cells. Cell lines containing a UCOE vector, showed a significantly higher and more consistent eGFP expression than the non‐UCOE cell lines without DHFR amplification. To further improve recombinant protein production cell lines were amplified with methotrexate (MTX). UCOE cell lines showed improved growth in MTX therefore amplification to 250 nM MTX was achieved following a one‐step amplification procedure. However, non‐UCOE cell lines showed higher levels of eGFP production following MTX amplification. In addition, UCOE cell lines did not improve stability during long‐term culture in the absence of selective pressure. Stable eGFP production was achieved for all cell lines when MTX is present. Finally, UCOE cell lines displayed more consistent response to external stimuli than non‐UCOE cell lines, suggesting that UCOE cell lines are less prone to clonal variability. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1014–1025, 2015     

Effects of dimethyl sulfoxide and dexamethasone on mRNA expression of housekeeping genes in cultures of C2C12 myotubes

We used quantitative real-time RT-PCR to investigate the effects of dimethyl sulfoxide (DMSO) and dexamethasone (Dex) on the mRNA expression levels of the housekeeping genes β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), phosphoglycerate kinase 1 (PGK1), peptidylprolyl isomerase A (PPIA), and transferrin receptor (TFRC) in cultures of C2C12 myotubes. The ratios of ACTB mRNA levels to the HPRT1 mRNA level in C2C12 cells that were differentiating from myoblast cells to myotubes decreased from 0 to 120 h of culture, whereas the ratios of TFRC mRNA levels to the HPRT1 mRNA level increased from 0 to 120 h of culture. The ratios of GAPDH, GUSB, PGK1, and PPIA mRNA levels to the HPRT1 mRNA level remained constant from 0 to 120 h of culture. All housekeeping gene mRNA levels were unaffected by exposure to DMSO concentrations of 0.1% or less. The GAPDH mRNA level was increased by Dex, while the ACTB and PGK1 mRNA levels were significantly decreased by Dex. The GUSB, PPIA, and TFRC mRNA levels were unaffected by exposure to Dex. GUSB, HPRT1, and PPIA are thus suitable internal controls for evaluating mRNA expression levels in cultures of C2C12 cells.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells treated in vitro with non-k-region dihydrodiols of 7-methylbenz[a]anthracene and benzo[a]pyrene

Studies were carried out on the incidence of sister-chromatid exchanges induced in Chinese hamster ovary cells by in vitro treatment with the polycyclic aromatic hydrocarbons 7-methylbenz[a]anthracene and benzo[a]pyrene and with related K-region and non-K-region dihydrodiols. Appreciable increases in the incidence of sister-chromatid exchanges were apparent in cells treated with non-K-region dihydrodiols: the most active compounds were 3,4-dihydro-3,4-dihydroxy-7-methylbenz[a]anthracene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and the effects were dose-dependent. The parent hydrocarbons and the related K-region dihydrodiols induced some sister-chromatid exchanges but they were considerably less active than these two non-K-region diols. The results suggest that this system may usefully be applied to studies aimed at determining which dihydrodiols are important in the metabolic activation of the carcinogenic polycyclic hydrocarbons. These and other results also infer that Chinese hamster ovary cells possess some intrinsic ability to metabolize such compounds in the absence of exogenous activation systems.     

长效人促卵泡激素CHO细胞株的构建及其体内活性

促卵泡激素(FSH)是具有促进卵泡与睾丸发育作用的一种垂体糖蛋白激素。因其体内半衰期较短,临床上需要连续10 d以上每天注射,病人使用非常不方便。本文旨在通过提高糖基化程度,研制一种长效FSH。通过一段含有两个N-糖基化位点的连接序列,将人FSHα链与β链cDNA融合,并插入pcDNA3.1(+)表达载体。表达载体转染CHO-K1细胞后,通过G418筛选得到阳性单克隆细胞,并经PCR和Western blotting证实。该重组FSH为单链蛋白,分子量约为49 kDa。经无血清培养,工程细胞株培养上清液中重组FSH的表达量可达3 mg/L。单次注射该重组FSH能够促进大鼠卵巢发育与卵泡成熟,且药效与连续8次注射Folltropin-V的相近。实验结果显示,本研究已成功获得一种长效重组FSH。     

Handling,processing and disposal of stem cell products in Europe: A survey by the cellular therapy and immunobiology working party of the European Society for Blood and Marrow Transplantation

Background

There is considerable heterogeneity in processing of stem cells for hematopoietic stem cell transplantation across Europe. The Foundation for the Accreditation of Cellular Therapy (FACT)–Joint Accreditation Committee International Society for Cellular Therapy and European Society for Blood and Marrow Transplantation (EBMT) (JACIE) standards provide minimum guidelines that, however, leave room for significant variations in practices at the individual transplantation center (TC).